Below are the draft of my comments on the EA of the GM pinkbollworm release in Arizona. APHIS allowed only 30 days for public comment and that is due about July 21. Hope others will send in comments to APHIS!

Environment assessment (EA) of the confined field study of a transgenic pink bollworm, Pectinophera gossypiella was prepared by US Department of Agriculture (USDA) , Animal and Plant Health Inspection Service (APHIS) to evaluate application for a permit application Jan. 17,2001 from APHIS. EA is designated Docket No. 01-024-1 and the comments below refer to that Docket.

Summary of the comments: The piggyBac transposon used to genetically modify pink bollworm was originally discovered by its ability to transpose within insect cells and baculovirus and to move between the two. The environment assessment of the pink bollworm release did not discuss the likelihood that baculovirus bearing piggyBac or related transposon could rescue the inactivated piggyBac in the pink bollworm cell by genetic recombination. The interaction of the piggyBac transposon in the bollworm cell and baculovirus should be studied in the laboratory before the "contained " field trial is allowed. The proposed trial does not "contain" the predictable interaction of baculovirus and pink bollworm. Baculovirus are known to cause non-pathological infection of human liver cells.

The objective of the proposed research associated with this permit application is to genetically engineer a strain of pink bollworm with an enhanced green fluorescent protein (GFP) marker gene derived from a jellyfish for field experimentation and performance studies. The use of a genetically marked insect in a PBW-SIT program would provide an additional tool for field managers to use in decisions involving efficient distribution of sterile PBW. The ability to identify the origin of native moth captures in the San Joaquin Valley of California is paramount to optimizing release strategies for this program. The multiple levels of physical and biological confinement in the proposed field tests are: (1) isolation by distance; (2) isolation by screen cages; (3) reproductive sterilization; (4) removing wings of females and placing them in secondary cages; (5) male pheromone traps; (6) destruction of the cotton that may contain bollworms; (7) flooding the area with a high-ratio of sterilized bollworms; and (8) insecticide treatment, if required.

The transgenic bollworms are genetically modified using the piggyback transposon. The piggyBac element is a deoxyribonucleic acid (DNA) transposable element capable of integrating into other DNA through mediation of a transposase encoded by a transposase Open Reading Frame (ORF) within the element, only when its Inverted Terminal Repeats (ITR) are intact. In the construct used for transformation of the PBW, the transposase gene of the piggyBac element was destroyed by insertion of an expression cassette containing EGFP ORF driven by a single copy of the Bombyx mori-derived BmA3 promoter. This manipulation destroys the ability of the transformation construct to move on its own.

Transformation was done by co-injecting a transposition and integration incompetent helper plasmid along with a donor plasmid into early stage PBW embryos. The donor plasmid contains the transforming construct flanked by piggyBac ITRs. The helper plasmid encodes an intact piggyBac transposase ORF. The gene product of this piggyBac transposase ORF is under the control of a promoter, that directs insect cells to express piggyBac transposase after injection. Importantly, the helper plasmid lacks the downstream piggyBac ITR. These ITRs are absolutely essential for piggyBac transposase mediated integration. Therefore, the helper plasmid lacking one or the other of the ITS cannot integrate itself into target DNA in a transposase-mediated event. The genes used from the donor organism and the piggyBac-derived portions of the vectors used to build the transforming construct were cloned off site. Specifically, Escherichia coli was the immediate host for the plasmids carrying the cloned genes used to make the transforming constructs. The piggyBac transposable element was discovered in cabbage looper cell culture at the University of Notre Dame (Fraser et al., 1995; Fraser et al., 1996; Wang and Fraser 1993).

The piggyBack vector was used to insert a green fluorescent protein into the genome of the bollworm. That protein is to be used as a marker to rapidly identify irradiated male moths used in the sterile moth control program. Later the piggyback vector may be used to insert female killing genes born by GM male moths to eradicate the bollworm pest. The green fluorescent marker poses no major threat to the environment should the gene escape but its incorporation into the cells of non-target organisms including human bears further study.

Even though piggyBack was discovered by its genetic impact on the baculovirus chromosome and its ability to move from insect to baculovirus (Fraser et al 1995,Fraser et al 1996, Wang and Fraser 1993) no laboratory experiments were submitted by APHIS to deal with the rescue of the inactivated piggyBac transposon by recombination with baculovirus. APHIS considered the potential rescue of inactivated piggyBac by recombination with related transposons in the bollworm genome and that possibility was dismissed. APHIS failed to consider the interaction of inactivated piggyBac with baculovirus in the environment of the field test. The field "containment" of the GM bollworm was totally uncontained regarding exposure of the GM bollworms to soil born baculovirus bearing active piggyBac transposon or transposons capable of rescueing the inactive GM piggyBac born by the bollworm. APHIS should have required extensive laboratory experiments using a range of baculovirus including strains with homologous GM piggyBac to provide good estimates of GM piggyBac escape through recombination or gene conversion mediated by baculovirus.

Rescue of the inactivated GM piggyBac transposon born by the pink bollworm by baculovirus bearing piggyBac or related transposon can be achieved by homologous recombination, gene conversion or by illegitimate recombination. APHIS seems to have ignored the obvious interaction between the soil born virus and the test bollworm and made no effort to contain or monitor the virus interaction. A strange exclusion based on the discovery of piggyBac by its genetic impact on baculovirus. The virus is normally soil born and can easily enter and leave the "containment" area proposed by APHIS as wind born dust or as clods on the shoes or gloves of workers. Certainly, APHIS bears the burden of proving the experiments to be safe and to protect the environment. We who comment do not have the means to evaluate the escapes that APHIS chooses to ignore.

Adequate laboratory studies must be done prior to the field release of potentially dangerous GM arthropods. Such experiments must include serious efforts to rescue the inactivated GM piggyBac using a range of baculovirus strains bearing piggyBac and baculovirus bearing transposons such as tagalong (Bauser et al 1999).

Ecological considerations for the impact of recombinant baculovirus insecticides have been studied extensively (Richards et al 1998). The study emphasized baculovirus containing scorpion toxin because that construction has been most widely studied. Impact on non-target insects is extrapolated from insects of related phylogeny, a practice difficult to defend. The recombinant baculovirus were very persistent and capable of reshaping an ecosystem. Modification of baculovirus host range specificity has been achieved by inserting or deleting genes (Theim 1997).

USDA has two patents related to baculovirus. Patent US6162430:Insect control with multiple toxins while US5639454:Recombinant baculovirus with broad host range. These patents may be relevant to the application on GM pink bollworm but do not explain why APHIS did not discuss baculovirus in the EA.

Baculovirus vectors efficiently transfer genes into human liver cells (Hofmann et al 1995; Boyce and Bucher 1996). The vectors transferred into human liver tissues most effectively in perfused liver tissue because serum components hampered virus transfer (Sandig et al 1996).Human conditions causing defects in complement should allow liver transfer of recombinant baculovirus. Inhibitors of complement facilitate baculovirus gene transfer (Hofmann and Strauss 1998). Hybrid baculovirus-adeno virus vectors have been used to deliver genes to human cells (Palombo et al 1998). Baculovirus vectors have beeen used to deliver hepatitis B to human liver efficiently to allow study of hepatitis B drug therapy (Delaney et al 1999).Recombinant piggyBac resued by baculovirus through recombination could infect humans with untoward consequences.

Baculovirus vectors are being used to control insect pests because they are effective and persist for a long time in the environment. Baculovirus vectors are also being used in gene therapy of human liver. These areas of research seem to exist as two solitudes and the risks of one are not evaluated in the context of the other.

APHIS noted that in the final analysis insecticide treatment could be used to eliminate all insect life from the test area. A tactical nuclear device might be required to deal with all of the ramifications of the field trial of the GM pink bollworm. Seriously, APHIS has frequently provided non-regulated status to GM crops that release pollen that fertilizes related wild species or nearby crops. This action is justified by the argument that herbicide treatment can eliminate nuisance products. That policy has begun to create problems. The argument that a pesticide drench can eliminate the mistakes of a poorly researched field release is not valid.

References

Bauser,C, Elick,T and Fraser,M "Proteins from nuclear extracts of two lepidopteran cell lines recongnize the ends of TTAA-specific transposons piggyBac and tagalong" 1999 Insect Mol Biol 8, 223-30.

Boyce,F and Bucher,N "Baculovirus-mediated gene transfer into mammalian cells" 1996Proc. Natnl Acad Sci USA 93, 2348-52.

Delaney,W, Miller,T, and Isom,H "Use of the hepatitis B virus recombinant baculovirus-Hep G2 system to study the effects of beta 2',3' dideoxy 3'thiaceydine on replication of hepatitis B virus and accumulation of covalently closed circular DNA"1999 Antimicrob Agents Chemother 43, 2017-26.

Fraser,M, Cary,L, Boonvisudhi,K and Wang,G " Assay for movement of lepidopteran transposon IFP2 in insect cells using a baculovirus genome as a target DNA."1995 Virology 211: 397-407.

Fraser,M, Ciszczon, T, Elick,T and Bauser, C " Precise excision of TTAA specific lepidopteran transposons piggyBac (IFP2) and tagalong (TFP3) from the baculovirus genome in cell lines from two species of Lepidoptera"1996 Insect Mol Biol 5,141-151.

Hofmann,C and Strauss,M "Baculovirus mediated gene therapy in the presence of human serum or blood facilitated by inhibition of the complement system" 1998 Gene Ther 5, 531-6.

Palombro,F, Mociotti,A, Recchia,A, Cortese,R, Ciliberto,G and LaMonica,N "Site specific integration in mammalian cells mediated by a new hybrid baculovirus-adeno-associated virus vector" 1998 J Virol 72, 5025-34.

Richards,A, Matthews,M and Christain,P "Ecological considerations for the environmental impact evaluation of recombinant baculovirus insecticides" 1998Ann Rev. Entomol 43, 493-517.

Sandig,V, Hofmann,C, Steinert,S, Jennings,G, Schlagg,P and Strauss,M "Gene transfer into hepatocytes and human liver tissue by baculovirus vectors" 1996 Human Gene Ther 20, 1937-45.

Thiem,S "Prospects for altering host range for baculovirus bioinsecticides" 1997 Curr Opin Biotechnol 8,317-22.

Wang, H., and J. Fraser. " TTAA serves as the target site for TFP3 lepidopteran transposon insertions in both nuclear polyhedrosis virus and Trichoplusia ni geneomes."1993 Insect Mol Biol (1): 109-16.